The plasma, or serine, proteases of the blood contact system are known to be activated by interaction with negatively charged surfaces. For example, tissue injury during surgery exposes the vascular basement membrane, causing interaction of the blood with collagen, which is negatively charged at physiological Ph. This induces a cascade of proteolytic events, leading to production of plasmin, a fibrinolytic protease, and consequent blood loss.
Perioperative blood loss of this type can be particularly severe during cardiopulmonary bypass (CPB) surgery, in which the patient's blood flow is diverted to an artificial heart-lung machine. CPB is an essential component of a number of life-saving surgical procedures. For example, in the United States, it is estimated that 300,000 patients every year undergo coronary artery bypass grafts involving the use of CPB.
Although necessary and generally safe, CPB is associated with a significant rate of morbidity, some of which may be attributed to a “whole body inflammatory response” caused by activation of plasma protease systems and blood cells through interactions with the artificial surfaces of the heart-lung machine (Butler et al., Ann. Thorac. Surg. 55:552 (1993); Edmunds et al., J. Card. Surg. 8:404 (1993)). For example, during extracorporeal circulation, exposure of blood to negatively charged surfaces of the artificial bypass circuit, e.g., plastic surfaces in the heart-lung machine, results in direct activation of plasma factor XII.
Factor XII is a single-chain 80 kDa protein that circulates in plasma as an inactive zymogen. Contact with negatively charged nonendothelial surfaces, like those of the bypass circuit, causes surface-bound factor XII to be autoactivated to the active serine protease factor XIIa. See Colman, Agents Actions Suppl. 42:125 (1993). Surface-activated factor XIIa then processes prekallikrein (PK) to active kallikrein, which in turn cleaves more XIIa from XII in a reciprocal activation reaction that results in a rapid amplification of the contact pathway. Factor XIIa can also activate the first component of complement C1, leading to production of the anaphylatoxin C5a through the classical complement pathway.
The CPB-induced inflammatory response includes changes in capillary permeability and interstitial fluid accumulation. Cleavage of high molecular weight kininogen (HK) by activated kallikrein generates the potent vasodilator bradykinin, which is thought to be responsible for increasing vascular permeability, resulting in edema, especially in the lung. The lung is particularly susceptible to damage associated with CPB, with some patients exhibiting what has been called “pump lung syndrome” following bypass, a condition indistinguishable from adult respiratory distress. See Johnson et al., J. Thorac. Cardiovasc. Surg. 107:1193 (1994).
Post-CPB pulmonary injury includes tissue damage thought to be mediated by neutrophil sequestration and activation in the microvasculature of the lung. (Butler et al., supra; Johnson, et al., supra). Activated factor XII can itself stimulate neutrophil aggregation. Factor XIIa-generated kallikrein, and complement protein C5a generated by Factor XIIa activation of the complement cascade, both induce neutrophil chemotaxis, aggregation and degranulation. See Edmunds et al., supra (1993). Activated neutrophils may damage tissue through release of oxygen-derived free-radicals, proteolytic enzymes such as elastase, and metabolites of arachidonic acid. Release of neutrophil products in the lung can cause changes in vascular tone, endothelial injury and loss of vascular integrity.
Intrinsic inhibition of the contact system occurs through inhibition of activated XIIa by C1-inhibitor (C1-INH). See Colman, supra. During CPB, this natural inhibitory mechanism is overwhelmed by massive activation of plasma proteases and consumption of inhibitors. A potential therapeutic strategy for reducing post-bypass pulmonary injury mediated by neutrophil activation would, therefore, be to block the formation and activity of the neutrophil agonists kallikrein, factor XIIa, and C5a by inhibition of proteolytic activation of the contact system.
Protease inhibitor therapy which partially attenuates the contact system is currently employed clinically in CPB. Aprotinin, also known as basic pancreatic protease inhibitor (BPPI), is a small, basic, 58 amino acid polypeptide isolated from bovine lung. It is a broad spectrum serine protease inhibitor of the Kunitz type, and was first used during bypass in an attempt to reduce the inflammatory response to CPB. See Butler et al., supra. Aprotinin treatment results in a significant reduction in blood loss following bypass, but does not appear to significantly reduce neutrophil activation. Additionally, since aprotinin is of bovine origin, there is concern that repeated administration to patients could lead to the development of an immune response to aprotinin in the patients, precluding its further use.
The proteases inhibited by aprotinin during CPB appear to include plasma kallikrein and plasmin. (See, e.g., Scott, et al., Blood 69:1431 (1987)). Aprotinin is an inhibitor of plasmin (Ki of 0.23 nM), and the observed reduction in blood loss may be due to inhibition of fibrinolysis through the blocking of plasmin action. Although aprotinin inhibits plasma kallikrein, (Ki of 20 nM), it does not inhibit activated factor XII, and consequently only partially blocks the contact system during CPB.
Another attractive protease target for use of protease inhibitors, such as those of the present invention, is factor XIIa, situated at the very first step of contact activation. By inhibiting the proteolytic activity of factor XIIa, kallikrein production would be prevented, blocking amplification of the contact system, neutrophil activation and bradykinin release. Inhibition of XIIa would also prevent complement activation and production of C5a. More complete inhibition of the contact system during CPB could, therefore, be achieved through the use of a better XIIa inhibitor.
Protein inhibitors of factor XIIa are known. For example, active site mutants of α1-antitrypsin that inhibit factor XIIa have been shown to inhibit contact activation in human plasma. See Patston et al., J. Biol. Chem. 265:10786 (1990). The large size and complexity (greater than 400 amino acid residues) of these proteins present a significant challenge for recombinant protein production, since large doses will almost certainly be required during CPB. For example, although it is a potent inhibitor of both kallikrein and plasmin, nearly 1 gram of aprotinin must be infused into a patient to inhibit the massive activation of the kallikrein-kinin and fibrinolytic systems during CPB.
The use of smaller, more potent XIIa inhibitors such as the corn and pumpkin trypsin inhibitors (Wen, et al., Protein Exp. & Purif. 4:215 (1993); Pedersen, et al., J. Mol. Biol. 236:385 (1994)) could be more cost-effective than the large α1-antitrypsins, but the infusion of high doses of these non-mammalian inhibitors could result in immunologic reactions in patients undergoing repeat bypass operations. The ideal protein XIIa inhibitor is, therefore, preferably, small, potent, and of human sequence origin.
One candidate for an inhibitor of human origin is found in circulating isoforms of the human amyloid β-protein precursor (APPI), also known as protease nexin-2. APPI contains a Kunitz serine protease inhibitor domain known as KPI (Kunitz Protease Inhibitor). See Ponte et al., Nature, 331:525 (1988); Tanzi et al., Nature 331:528 (1988); Johnstone et al., Biochem. Biophys. Res. Commun. 163:1248 (1989); Oltersdorf et al., Nature 341:144 (1989). Human KPI shares about 45% amino acid sequence identity with aprotinin. The isolated KPI domain has been prepared by recombinant expression in a variety of systems., and has been shown to be an active serine protease inhibitor. See, for example, Sinha, et al., J. Biol. Chem. 265:8983 (1990). The measured in vitro Ki of KPI against plasma kallikrein is 45 nM, compared to 20 nM for aprotinin.
Aprotinin, KPI, and other Kunitz-type serine protease inhibitors have been engineered by site-directed mutagenesis to improve inhibitory activity or specificity. Thus, substitution of Lys15 of aprotinin with arginine resulted in an inhibitor with a Ki of 0.32 nM toward plasma kallikrein, a 100-fold improvement over natural aprotinin. See PCT application No. 89/10374. See also Norris et al., Biol. Chem. Hoppe Seyler 371:3742 (1990). Alternatively, substitution of position 15 of aprotinin with valine or substitution of position 13 of KPI with valine resulted in elastase inhibitors with Kis in the 100 pM range, although neither native aprotinin nor native KPI significantly inhibits elastase. See Wenzel et al., in: Chemistry of Peptides and Proteins, Vol. 3, (Walter de Gruyter, Berlin, N.Y., 1986); Sinha et al., supra. Methods for substituting residues 13, 15, 37, and 50 of KPI are shown in general terms in European Patent Application No. 0 393 431, but no specific sequences are disclosed, and no protease inhibition data are given.
Phage display methods have been recently used for preparing and screening derivatives of Kunitz-type protease inhibitors. See PCT Application No. 92/15605, which describes specific sequences for 34 derivatives of aprotinin, some of which were reportedly active as elastase and cathepsin inhibitors. The amino acid substitutions in the derivatives were distributed throughout almost all positions of the aprotinin molecule.
Phage display methods have also been used to generate KPI variants that inhibit factor VIIa and kallikrein. See Dennis et al., J. Biol. Chem. 269:22129 and 269:22137 (1994). The residues that could be varied in the phage display selection process were limited to positions 9-11, 13-17, 32, 36 and 37, and several of those residues were also held constant for each selection experiment. One of those variants was said to have a Ki of 1.2 nM for kallikrein, and had substitutions at positions 9 (Thr→Pro), 13 (Arg→Lys), 15 (Met→Leu), and 37 (Gly→Tyr). None of the inhibitors was tested for the ability to inhibit factor XIIa.
It is apparent, therefore, that new protease inhibitors that can bind to and inhibit the activity of serine proteases are greatly to be desired. In particular it is highly desirable to prepare peptides, based on human peptide sequences, that can inhibit selected serine proteases such as kallikrein; chymotrypsins A and B; trypsin; elastase; subtilisin; coagulants and procoagulants, particularly those in active form, including coagulation factors such as factors VIIa, IXa, Xa, XIIa, and XIIa; plasmin; thrombin; proteinase-3; enterokinase; acrosin; cathepsin; urokinase; and tissue plasminogen activator. It is also highly desirable to prepare novel protease inhibitors that can ameliorate one or more of the undesirable clinical manifestations associated with enhanced serine protease activity, for example by reducing pulmonary damage or blood loss during CPB.